Use of CultureSwab Plus Swabs with Amies Gel Agar with the IDI-MRSATM Assay

J. Riley, B. Shoemaker, K. Jones, and P. Bourbeau Geisinger Medical Center, Danville, PA. 17822

C-106

REVISED ABSTRACTThe IDI-MRSATM assay (GeneOhm Sciences, San Diego,CA) is an FDA-cleared test for the direct detection ofnasal colonization with methicillin-resistantStaphylococcus aureus (MRSA). Many institutions,including ours, selectively or broadly screen new admis-sions for MRSA nasal colonization to facilitate appropriateisolation procedures for MRSA carriers. Utilized in con-junction with the Smart CyclerTM instrument (Cepheid,Sunnyvale, CA), the IDI-MRSATM assay provides rapidreal-time results. The product insert specifies that speci-mens to be tested with the IDI-MRSATM assay be collect-ed with a swab with liquid Stuart media such as the BBLCultureSwab. In our laboratory, we routinely utilize theBBL CultureSwab Plus, an Amies gel swab collectiondevice. The purpose of this study was to compare the useof the CultureSwab and the CultureSwab Plus to detectMRSA using the IDI-MRSATM assay. The standard IDI-MRSATM protocol used for swabs with liquid Stuart mediawas modified slightly for use with agar gel swabs with theaddition of one extra processing step, heating at 95 +/-2C for 2 minutes. The remainder of the processing wasidentical to the protocol used for swabs with liquid media.Preliminary work in our laboratory with the IDI-MRSATM

assay indicated that the sensitivity of the assay was gen-erally between 102 and 103 CFU. For this evaluation, weprepared dilutions with intended final concentrations of100, 500, and 1000 CFU that were used to inoculateCultureSwab swabs with liquid Stuart’s media andCultureSwab Plus swabs with Amies gel agar media. Atotal of 6 swabs were inoculated for each isolate (2 mediaat 3 dilutions).120 recent clinical isolates were tested with274 out of a possible 360 positive test results with theCulture Swab Plus swabs with Amies gel and 303 out ofa possible 360 positive test results for the CultureSwabswabs with liquid Stuart’s media (p=0.0003). Theseresults suggest that the CultureSwab Plus swabs withAmies gel are an acceptable alternative to theCultureSwab swabs with liquid Stuart’s media for use inthe IDI-MRSATM assay but may not be as sensitive whenspecimens contain lower concentrations of organisms.

CONCLUSIONS• Testing of gel swabs with the IDI-MRSATM assay requires only a

minor modification of the protocol used for the liquid swabs.• The liquid swab is more sensitive than the gel swab for detection

of MRSA when lower concentration of inocula are used.• The clinical significance of the differences in vitro sensitivity is

not known. We stressed the assay/swab system by testing atinocula concentrations close to the limits of sensitivity of the IDI-MRSATM assay.

MATERIALS AND METHODSI. 120 recent unique patient clinical isolates of

Staphylococcus aureus were used for this study. II. Dilutions were made to prepare 3 inocula with targeted

concentrations of 10(4), 5 x 10(3) and 10(3) per ml. III. 0.1 ml was added to each swab. A total of 6 swabs were

inoculated for each isolate (2 media at 3 dilutions) usinga microtiter plate. Final targeted inocula were 100, 500,and 1000 CFU/swab.

IV.Processing. Liquid Stuart gel swabs were processedfollowing instructions in the package insert. For the agargel swabs, an additional step was added (indicated inblack below):A. Remove one swab from the holder and place in the

sample buffer tube. Break the swab stem off into thetube. Do this by holding the swab stem near the rimof the tube (use a kim-wipe to minimize the risk ofcontamination). Lift the swab a few millimeters fromthe bottom and bend the stem against the tube tobreak it. Discard the swab stem and kim-wipe.Tightly cap the sample buffer tube.

B. Vortex at high speed for one minute.C. Using a sterile disposable fine-tip pipet, transfer the

entire cell suspension to a yellow-capped lysis tube.D. Heat at 95 +/- 2C for 2 minutes.E. Centrifuge at high speed (>14000 x g) for 5 minutes

at room temperature.F. Using a sterile disposable fine-tip pipet, remove the

supernatant and discard. Be careful not to touch thepellet during this step.

G. Add 50 ul of sample buffer to each lysis tube andclose tightly. Use a new pipette tip for each speci-men. Save remaining buffer.

H. Vortex at high speed for five minutes.I. Centrifuge the lysis tubes briefly using the quick spin

button on the centrifuge. Hold for 8 seconds andthen release.

J. Heat the lysis tubes at 95 +/- 2C for 2 minutes.K. Remove the lysis tubes and place in tube holder and

maintain at 2-8C until pipetted into the Smart Cyclerreaction tubes.

TABLE 1Number of Positive Test Results for each swab type with different con-centrations of inocula

No of Positive Resultsa

Concentration Both Gel Swab Liquid Swab P value

1,000 CFU 117 0 3 NSb

500 CFU 98 4 16 0.007

100 CFU 40 15 29 0.03

Total 255 19 48 0.0003

a maximum of 120 positive results for each concentrationb Not statistically significant